Project Summary. Loss of endothelial barrier integrity is central to pathogenesis of acute respiratory distress syndrome (ARDS), a severe lung disease, associated with sepsis and trauma. Thus, homeostatic replenishment of ECs after injury is required for the efficient formation of a stable endothelial barrier and to prevent long-lasting lung vascular injury but the identity and source of these ?reparative endothelial cells (ECs)? remains unclear. Given the findings that ECs can convert into fibroblast-like cells (FLC) upon tissue injury and that forced expression of the ETS family of transcription factors (e.g., ETV2) reprogram FLC into ECs, a fundamental unanswered question is whether a shift from the ?reparative? EC lineage into a non-reparative FLC lineage impairs endothelial regeneration and thereby recovery from lung vascular injury. PTEN (Phosphatase and TENsin homolog) is a well-known tumor suppressor and regulates key features of vascularization such as endothelial migration and proliferation. Our Preliminary Data, showed that EC-specific conditional deletion of PTEN markedly decreased lung EC regeneration while FLCs were increased and these mice developed lung edema spontaneously. Further experiments showed that PTEN localized to the nucleus of ECs and maintained the expression of endothelial transcription factor, Ets-Related Gene (ERG). Based on these intriguing Preliminary Data, and using a range of powerful approaches such as novel genetic mouse models (inducible dual-reporter EC-PTEN and EC-ERG null mice for lineage tracing), RNAseq and measurements of lung vascular permeability we will investigate the hypothesis that PTEN-induced ERG pathway is a key determinant of the EC lineage and resolution of lung-fluid balance. Our Specific Aims are: #1: to investigate the hypothesis that EC-expressed PTEN drives endothelial cell regeneration at the expense of fibroblast-like cells and thereby maintains the lung vascular barrier; #2: to determine (a) the role of the endothelial transcription factor, ERG, as an effector of PTEN and a key determinant of EC lineage, and (b) to identify ERG enriched endothelial cells through endothelial lineage tracing as intrinsic reparative ECs whose activation promotes lung vascular regeneration and restoration of lung fluid balance. With these comprehensive studies, we will identify for the first time the role of the PTEN-ERG cascade in maintaining the reparative EC lineage and in resolving inflammatory lung vascular injury. We expect our studies to lay a new conceptual framework for the development of novel therapeutic approaches exploiting PTEN as a key target for the treatment of ARDS.